Proper organization of the microtubule cytoskeleton underlies many cellular functions such as polarized transport in neurons, nutrient transport in epithelial cells, and mitosis. While mechanisms that control microtubule alignment in mitotic cells have been extensively studied, alignment of microtubules in differentiated cells has been probed much less. Dendrites of Drosophila neurons can be used as a system to identify mechanisms that control microtubule polarity as they contain uniform-polarity minus-end-out microtubules. In vivo experiments in fly neurons have led to a model of microtubule alignment in dendrites in which a complex of kinesin-2 and plus-tip interacting proteins (+TIPs) at growing microtubule plus-ends interacts with stationary microtubules at branch points and actively directs the growing plus- end toward the cell body. This microtubule steering mechanism may serve as a general model for control of microtubule polarity in diverse cell types such as epithelial cells. The goal of this proposal is to use in vitro reconstitution, computational simulations, and analysis of Drosophila neurons to understand +TIP-kinesin based microtubule steering. The first aim of the work is to use purified proteins and microfabricated channels to develop a novel experimental system for studying microtubule steering by +TIP-kinesin complexes in vitro. This in vitro reconstitution will validate and extend in vivo observations, and will provide a system for quantifying the activity of this protein complex. The second aim will be to measure binding affinities between specific components of the system to establish quantitative biochemical parameters for modeling studies. The third aim will be to develop computational simulations of +TIP-kinesin based microtubule steering in vivo and in vitro, using quantitative parameters generated from the experiments, and then test predictions of the models using in vivo experiments. The simulations will incorporate known mechanical properties of microtubules and kinesin motors, and will provide insights into the ability of +TIPs, to withstand the mechanical loads necessary for sustained microtubule bending. These experimental and computational studies will explore novel functional roles for both kinesin motors and the +TIP proteins, and the framework developed here will provide a foundation for understanding universal aspects of microtubule polarity establishment in differentiated cells. The importance of understanding proper microtubule organization in neurons is underscored by the numerous human neurodegenerative diseases that are linked to mutations in genes involved in regulating the microtubule cytoskeleton. PUBLIC HEALTH RELEVANCE: Microtubule-based transport is crucial for the growth and maintenance of neurons and transport deficiencies are linked to neurodegenerative diseases such as ALS, Alzheimers and Huntington's disease. Furthermore, following axon removal, kinesin-2 has been shown to be required for proper regrowth of an axon from a dendrite, suggesting that the microtubule steering mechanism examined here may play a role in the repair of neurons from trauma. Thus, insights from this work could be applied to enhancing neural regeneration following injury in humans.